ENDOCRINE REGULATIONS, Vol. 32, 63 – 70, 1998

63

LONG-TERM EFFECTS OF VARIOUS IODINE AND FLUORINE DOSES ON THE THYROID AND FLUOROSIS IN MICE W. ZHAO1,2, H. ZHU1, Z. YU1, K. AOKI2, J. MISUMI2, X. ZHANG3 Department of Environmental Hygiene, Hebei Medical University, Shijiazhuang 050017, China; 2Department of Public Health and Hygiene, Oita Medical University, Oita 879-5593, Japan; 3Department of Pathology, Hebei Medical University, Shijiazhuang 050017, China

1

Objective. To elucidate the participation of the independent and combined long term effect of various concentrations of iodine and fluorine on the pathogenesis of goiter and fluorosis in mice. Methods. Nine drinking water supplies with different iodine and fluorine content were prepared by combination of potassium iodate and sodium fluoride solutions in bidistilled water. The concentrations of iodide were: 1. iodine deficiency (ID): 0.0; 2. iodine normal (IN): 20.0; 3. iodine excess (IE) 2500.0 µg/l; and these of fluoride were: 1. fluoride deficiency (FD) 0.0; 2. fluoride normal 0.6; 3. fluoride excess (FE), 30.0 mg/l. A total of 288 Kunmim mice was divided into 9 groups consisting of 32 animals each and each group, in addition to basal diet, received one of following iodide/fluoride mixtures: ID+FD, ID+FN, ID+FE, IN+FD, IN+FN, IN+FE, IE+FD, IE+FN, IE+FE. By such manner, one half of the animals of each group was treated for 100 days and the other half for 150 days. Results. It was found that ID only and IE only could both induce the goiter. FE induced dental fluorosis and increased fluorine content in the bone. In addition, fluorine also affected the thyroid changes induced by ID or IE. After 100 days of treatment, fluorine showed some stimulatory effect on the thyroid in ID conditions and inhibitory effect in IE conditions. After 150 days, however, the effects of fluorine on the thyroid reversed as compared with that of 100 days. On the other hand, difference of iodide intake could also increase the toxic effects of FE on the incisors and bones. The rate and degree of the incisor fluorosis, the fluorine contents in the bone were significantly higher in the ID+FE group than those in the IN+FE and IE+FE groups. Conclusions. Both iodine deficiency and excess induced goiter as well as other functional and histopathological changes in the mouse thyroid. Excessive fluorine caused fluorosis of incisors and limb bones. In addition, iodine and fluorine do have mutually interacting effects on both goiter and fluorosis in the experimental mice. Key words: Iodine – Fluorine -Various doses – Goiter- Fluorosis – Mice

The distribution of iodine and fluorine in the environment, especially in the underground water, is usually similar. Yet, endemic goiter (iodine deficiency disorder – IDD) and fluorosis seldom occur in the same area or in the same population at the same time (FENG 1981; LIN 1984). However, even the excess of iodine in water and food has been shown to induce goiter both in normal and in endemic fluorosis coastal and inland areas in China and possible effects of fluorine on goiter were noticed (MA et al. 1982; ZHU et

al. 1984; YU et al. 1988; YANG et al. 1994). In addition to fluorine present in underground waters, even the fluorine pollution from burning coal appears to be another important pathogenic factor of the endemic fluorosis in China (LI 1982). Thus, there is no doubt that some rural IDD areas in China may be equally affected by fluorine pollution from coal burning. Actually, even the fluorosis caused by excess fluorine in drinking water was also reported in one area of IDD (REN et al. 1989).

64

EFFECTS OF IODINE AND FLUORINE ON GOITER & FLUOROSIS

So far, the observations on the exact effects of fluorine on the thyroid have been quite controversial (DAY 1972; BOBEK et al. 1976; HARA 1980; BAUM et al. 1981; SIEBENHURER et al. 1984; YU et al. 1985; ZHAO et al. 1988, 1992; YANG et al. 1994). One of possible reasons for this is thought to be the different iodine intake interfering with the effect of fluorine on thyroid (DEMOLE 1970; BUERGI et al. 1984). Thus, it appears reasonable to study the effects of iodine and fluorine on the thyroid under various combinations of iodine and fluorine intake as well as the iodine influence on fluorosis induced by fluorine. It was found that the response to iodine and fluorine excess in mouse resembles that found in man (ZHU et al. 1988; ZHAO et al. 1992). Thus, the aim of this study was to elucidate both the independent and combining effects of those two elements on the pathogenesis of goiter and fluorosis. Materials and Methods Experimental animals and treatment. A total of 288 male Kunmin mice weighing 13 to 15 g was purchased from the Experimental Animal Institute, Chinese Medical Academy. After adaptation, they were randomly divided into nine groups of 32 animals each (from which sick animals that suffered a infectious disease after long-term study were excluded for experimental observation) and were allowed free access to basal chow and drinking water with different amounts of iodine and fluorine (see bellow). The iodine and fluorine solutions were prepared by the addition potassium iodate (KIO3) and/or sodium fluoride (NaF) into bidistilled water. Three concentrations of iodine (iodine deficiency [ID] – 0.0; iodine normal [IN] – 20.0 and iodine excess [IE] – 2500 µg/l and fluorine (fluorine deficiency [FD] – 0.0, fluorine normal [FN] – 0.6 and fluorine excess [FE] – 30.0 mg/l) were used. Special low iodine and low fluorine chow was prepared by the Experimental Animal Institute of Hebei Medical University with the use of grains from an IDD area in China: corn 38 %, wheat 33 %, millet grain 20 %, soybean 7 %, yeast 1 %, soybean oil 0.5 %, sodium chloride 0.5 %, egg 1 %. The body weight of all animals was estimated in two week intervals. Ethical introduction contained in “Guidelines on the Handling and Training of Laboratory Animals” were observed.

Evaluation of fluorosis. Two weeks after the beginning of experiment, incisor fluorosis was checked weekly and classified using the criteria shown in Tab. 1. Radioiodine uptake. After 100 and 150 days of treatment the carrier containing 131I was injected i.p. in a dose of 0.2 µCi/100 g. The animals were sacrificed 6 h later, the blood was withdrawn and the thyroids were dissected. The 131I uptake by the thyroid was estimated routinely. Histopathological observations. The thyroids were weighed and fixed in 10 % formol, embedded in paraffin and the sections of 5 µm thickness were stained with hematoxyline and eosine. The follicular diameter and follicular cell height were measured in the light microscope. According to histological findings the thyroid was classified as hyperplastic goiter (HG), colloid goiter (CG), normal or almost normal thyroid (NT), intermediate status between NT and HG (abbreviated as NH) or between NT and CG (abbreviated as NC). Estimation of serum triiodothyronine (T3) and thyroxine (T4). The sera obtained by centrifugation were stored at -30 oC until assayed. Serum triiodothyronine and thyroxine were estimated by commercial kits purchased from the General Navy Hospital (Beijing, China). Estimation of fluorine in bones, fluorine and iodine in drinking water and chow. The selective fluoride electrode method was used for the estimation of fluoride in the ashes of the limb bones (recovery 84.9 to 110.76 %), drinking water and in the mouse chows (recovery 89.9 to 105.27 %). The mouse chow has been burned at 550 oC for 3 h, and iodine in the ashes has been extracted with bidistilled water. The content of iodine in the water extracted from the ashes and the drinking water for the nine groups of mice was analyzed by the Ce/As method (recovery 86.81 – 96.75 %). Statistical evaluation. ANOVA and ONE WAY in a statistical software named SPSS (NORUSIS 1995) and the “Cross Product Different Method (CPD)” (WANG 1981) were used to analyze the qualitative and ranked data, respectively. With these methods either the effect of iodine or fluorine alone or the combined effects of iodine and fluorine can be analyzed. The differences between every pair of two groups were also analyzed after the significant difference among the whole 9 groups has been found with the methods described above.

65

EFFECTS OF IODINE AND FLUORINE ON GOITER & FLUOROSIS Tabl e 1 Criterion of Incisor fluorosis Normal

Questionable

Mild

Moderate

Severe

Glossy

Good

Good

Not good

Bad

Bad

Color

Deep yellow of upper incisor or Egg white color of lower incisor

Light yellow of upper incisor and white lower incisor

Deep white of of lower incisor

Deep white of lower incisor

Deep white of lower incisor

semivitriform

Slight Aberration of translucency

Opaque

Opaque

Opaque

Chalky white area

No

No

Flecks or spots

Areas

Areas

Pit or break

No

No

No

No

Have

Vitrifiction

Tabl e 2 Changes in Body Weight of Mice after 100 and 150 Days (g) Fluorine Level

Iodine level IN

IE

Significant for iodineb

FD

a

31.5±6.76 (11) 49.7±2.91 (12)

35.0±4.15 (14) 51.9±5.13 (14)

34.1±5.36 (16) 49.9±4.98 (9)

P>0.05 P>0.05

FN

32.2±6.20 (11) 50.7±4.71 (9)

40.7±6.12 (13) 56.0±1.41 (8)

39.3±5.45 (7) 55.4±5.31 (14)

P>0.01 P>0.05

FE

34.1±3.91 (12) 51.5±4.12 (14)

38.5±3.92 (16) 53.5±2.46 (12)

39.5±4.95 (12) 53.7±3.83 (15)

P>0.01 P>0.05

P>0.05 P>0.05

P0.05

P